Iron is an essential trace element required for growth and development of all living organisms. Iron content in mammals is regulated by controlling iron absorption, iron recycling, and release of iron from the cells in which it is stored. Iron is absorbed predominantly in the duodenum and upper jejunum by enterocytes. A feedback mechanism exists that enhances iron absorption in individuals who are iron deficient, and that reduces iron absorption in individuals with iron overload (Andrews Ann. Rev. Genomics Hum. Genet. 1:75 (2000); Philpott, Hepatology 35:993(2002); Beutler et al., Drug-Metab. Dispos. 29:495(2001)). Iron is recycled from degraded red cells by reticuloendothelial macrophages in bone marrow, hepatic Kupffer cells and spleen. Iron release is controlled by ferroportin, a major iron export protein located on the cell surface of enterocytes, macrophages and hepatocytes, the main cells capable of releasing iron into plasma. Hepcidin binds to ferroportin and decreases its functional activity by causing it to be internalized from the cell surface and degraded. (Nemeth et al., Science, 306:2090-3, 2004; De domenico et al., Mol. Biol. Cell., 8:2569-2578, 2007).
Hepcidin is the key signal regulating iron homeostasis (Philpott, Hepatology 35:993 (2002); Nicolas et al., Proc. Natl. Acad. Sci. USA 99:4396 (2002)). High levels of human hepcidin result in reduced iron levels, and vice versa. Mutations in the hepcidin gene which result in lack of hepcidin activity are associated with juvenile hemochromatosis, a severe iron overload disease (Roetto et al., Nat. Genet., 33:21-22, 2003). Studies in mice have demonstrated a role of hepcidin in control of normal iron homeostasis (Nicolas et al., Nat. Genet., 34:97-101, 2003; Nicolas et al., Proc. Natl. Acad. Sci. USA, 99:4596-4601, 2002; Nicolas et al., Proc. Natl. Acad. Sci. USA, 98:8780-8785, 2001.).
In addition, data is accumulating implicating hepcidin in iron sequestration during inflammation (See, e.g., Weinstein et al., Blood, 100:3776-36781, 2002; Kemna et al., Blood, 106:1864-1866, 2005; Nicolas et al., J. Clin. Invest., 110:1037-1044, 2002; Nemeth et al., J. Clin. Invest., 113:1271-1276, 2004; Nemeth et al., Blood, 101:2461-2463, 2003 and Rivera et al., Blood, 105:1797-1802, 2005). Hepcidin gene expression has been observed to be robustly upregulated after inflammatory stimuli, such as infections, which induce the acute phase response of the innate immune systems of vertebrates. In mice, hepcidin gene expression was shown to be upregulated by lipopolysaccharide (LPS), turpentine, Freund's complete adjuvant, and adenoviral infections. Hepcidin expression is induced by the inflammatory cytokine interleukin-6 (IL-6). A strong correlation between hepcidin expression and anemia of inflammation was also found in patients with chronic inflammatory diseases, including bacterial, fungal, and viral infections.
Human hepcidin, a 25 amino acid peptide with anti-microbial and iron-regulating activity, was discovered independently by two groups investigating novel anti-microbial peptides. (Krause et al., FEBS Lett. 480:147 (2000); Park et al., J. Biol. Chem. 276:7806 (2001)). It has also been referred to as LEAP-1 (liver-expressed antimicrobial peptide). A hepcidin cDNA encoding an 83 amino acid pre-propeptide in mice and an 84 amino acid pre-propeptide in rat and human were subsequently identified in a search for liver specific genes that were regulated by iron (Pigeon et al., J. Biol. Chem. 276:7811 (2001)). The 24 residue N-terminal signal peptide is first cleaved to produce pro-hepcidin, which is then further processed to produce mature hepcidin, found in both blood and urine. In human urine, the predominant form contains 25 amino acids, although shorter 22 and 20 amino acid peptides are also present.
The mature peptide is notable for containing eight cysteine residues linked as four disulfide bridges. The structure of hepcidin was studied by Hunter et al., J. Biol. Chem., 277:37597-37603 (2002), by NMR using chemically synthesized hepcidin with an identical HPLC retention time to that of native hepcidin purified from urine. Hunter et al. reported their determination that hepcidin folded into a hairpin loop structure containing a vicinal disulfide bond (C1-C8, C2-C7, C3-C6, C4-C5). More recently, determination of the structure of bass hepcidin was also reported, using the structural information of Hunter et al. and inferential NMR data to deduce an identical disulfide connectivity assignment (Lauth et al., J. Biol. Chem., 280:9272-9282 (2005). However, as discovered and disclosed herein by the present inventors, the structure of hepcidin was determined to have a disulfide bond connectivity that is different from that taught by the prior art.
U.S. Patent Publication Nos. 2003/0187228, 2004/0096987, 2004/0096990, 2005/0148025, 2006/0019339, 2005/0037971 and 2007/0224186; U.S. Pat. Nos. 7,232,892 and 7,294,690 and International Publication No. WO 02/98444 discuss hepcidin antibodies but fail to disclose or suggest the structural conformation of hepcidin disclosed herein.
Thus, the specification illustrates the determination of the structure of hepcidin, as well as the central role of hepcidin and its key functions in iron regulation and in the innate immune response to infection. Furthermore, the application provides, inter alia, bioactive hepcidin, monoclonal antibodies to the bioactive hepcidin, methods to produce the same, methods to determine bioactive hepcidin, and methods to modulate hepcidin activity or its expression, and methods for treating disorder of iron homeostasis as well as hepcidin antagonists and hepcidin agonists.